International Journal of Nanomedicine

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Cytoprotective and enhanced anti-inflammatory activities of liposomal piroxicam formulation in lipopolysaccharide-stimulated RAW 264.7 macrophages
This article was published in the following Dove Press journal: International Journal of Nanomedicine 22 March 2013 Number of times this article has been viewed

Hoe Siong Chiong 1 Yoke Keong Yong 1 Zuraini Ahmad 1 Mohd Roslan Sulaiman 1 Zainul Amiruddin Zakaria 1 Kah Hay Yuen 2 Muhammad Nazrul Hakim 1,3
Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia; 2School of Pharmaceutical Sciences, Universiti Sains Malaysia, Gelugor, Malaysia; 3Sports Academy, Universiti Putra Malaysia, Serdang, Malaysia
1

Background: Liposomal drug delivery systems, a promising lipid-based nanoparticle technology, have been known to play signicant roles in improving the safety and efcacy of an encapsulated drug. Methods: Liposomes, prepared using an optimized proliposome method, were used in the present work to encapsulate piroxicam, a widely prescribed nonsteroidal anti-inammatory drug. The cytotoxic effects as well as the in vitro efcacy in regulation of inammatory responses by free-form piroxicam and liposome-encapsulated piroxicam were evaluated using a lipopolysaccharide-sensitive macrophage cell line, RAW 264.7. Results: Cells treated with liposome-encapsulated piroxicam demonstrated higher cell viabilities than those treated with free-form piroxicam. In addition, the liposomal piroxicam formulation resulted in statistically stronger inhibition of pro-inammatory mediators (ie, nitric oxide, tumor necrosis factor-, interleukin-1, and prostaglandin E2) than piroxicam at an equivalent dose. The liposome-encapsulated piroxicam also caused statistically signicant production of interleukin-10, an anti-inammatory cytokine. Conclusion: This study afrms the potential of a liposomal piroxicam formulation in reducing cytotoxicity and enhancing anti-inammatory responses in vitro. Keywords: liposomes, nitric oxide, cytokines, prostaglandin E2, interleukin-1, piroxicam

Introduction
Macrophages play a critical role in the regulation of inammation and immune responses that protect the host against microbial invasion as well as tissue injury.1,2 Lipopolysaccharide (LPS), a well-studied component from the outer membrane of Gram-negative bacteria, is widely considered one of the most potent activators of macrophages.3,4 During an inammatory process, activated macrophages initiate a diverse series of functional responses such as the production of nitric oxide (NO) and cytokines (eg, tumor necrosis factor [TNF], interleukin [IL], and growth factors) as well as the activation of phospholipase A2, which produces lipid metabolites of arachidonic acid (eg, prostaglandin [PG] and leukotrienes).57 Recognition of bacterial LPS endotoxin and subsequent intracellular signal transduction cascades in macrophages are key in eliminating invading pathogens and deleterious stimuli.8,9 Nevertheless, an excessive or continuous production of inammatory mediators has been linked to the development of various acute and chronic human diseases, including septic shock, hemorrhagic shock, atherosclerosis, rheumatoid arthritis, ulcerative

Correspondence: Muhammad Nazrul Hakim Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Tel +60 3 8947 1133 Fax +60 3 8947 1126 Email nazrul.hakim@gmail.com

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http://dx.doi.org/10.2147/IJN.S42801

International Journal of Nanomedicine 2013:8 12451255 1245 2013 Chiong etal, publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.

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colitis, multiple sclerosis, hepatitis, pulmonary brosis, and cancer. Hence, an adequate inhibition of these mediators is essential in suppressing enduring inammatory process and preventing inammation-driven diseases.10,11 In the present work, the in vitro efcacy of different drug formulations (ie, free form and liposome based) in regulating inammatory responses by inhibition of different inammatory mediators was assessed using a sensitive LPS-stimulated macrophage model. Liposomal encapsulation technology has been demonstrated in various studies to ameliorate the therapeutic indices and pharmacological activities of conventional drug formulations.12,13 In addition, the lipid-based nanoparticle drug carrier is becoming increasingly popular due to its successes in altering the biopharmacological properties of entrapped hydrophobic drugs (eg, improving the drugs solubility, dissolution kinetics, and bioavailability).1416 Hence, utilization of this promising liposomal delivery system to enhance the pharmacological properties exhibited by piroxicam in a cellular model of inammation is truly worthwhile. Piroxicam, an oxicam derivative, is among the most frequently prescribed nonsteroidal anti-inflammatory drugs (NSAIDs) for the management of inflammation in various musculoskeletal diseases.17,18 As with other NSAIDs, piroxicams mechanism of action is not completely understood. It is generally agreed that its therapeutic properties are primarily derived from the decreased formation of PG precursors.18,19 Owing to its large market potential, a feasible strategy for maximizing the drugs benets to patient health while minimizing its toxic side effects is, therefore, highly desired.

Preparation of liposome samples

Pro-lipo Duo, a commercially available proliposome mixture, was used to prepare piroxicam-loaded and blank liposomal samples in accordance with an optimized procedure previously described.20 Briey, stock piroxicam solution (60mg/mL DMSO) was added into Pro-lipo Duo and stirred moderately (12525 rpm) for 1hour. Concentrated piroxicam-loaded liposomal suspension was formed by the dropwise addition of distilled water (dH2O). This liposomal suspension was hydrated by 10hours of continuous stirring at room temperature before being further diluted with dH2O and stirred continuously for another 30minutes. The ratio of stock piroxicam solution to Pro-lipo to dH2O (hydration) to dH2O (dilution) was 1:5:9:25 w/w/w/w. Blank liposomes were prepared following the same procedure, except that DMSO was used instead of stock piroxicam solution. All freshly prepared samples were diluted to the required drug concentration and volume before use.

Characterization
The drug entrapment and size proles of the prepared liposomal samples were determined using high-performance liquid chromatography (Jones HPLC Genesis C18 column, Biotage, Uppsala, Sweden) and photon correlation spectroscopy (Zetasizer Nano S, Malvern Instruments, Malvern, UK), respectively, as previously reported.20,21 Duplicate samples for analysis were prepared from each of the three individual batches of liposomal samples (n = 6). The morphological observation of blank liposomes and liposome-encapsulated piroxicam was done using a Philips CM12 transmission electron microscope (Amsterdam, The Netherlands).

Materials and methods Materials

Pro-lipo Duo was obtained from Lucas Meyer Cosmetics (Champlan, France). Piroxicam, dimethyl sulfoxide (DMSO), LPS from Escherichia coli (serotype 055:B5, phenol extract), and phosphate-buffered saline were purchased from SigmaAldrich (St Louis, MO, USA). The RAW 264.7macrophage cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbeccos modified Eagles medium (DMEM), fetal bovine serum, and penicillinstreptomycin solution were purchased from Thermo Fisher Scientic (Waltham, MA, USA), while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from EMD Millipore (Billerica, MA, USA). Griess reagent was purchased from Merck (Darmstadt, Germany).

Cell culture and treatment

RAW 264.7macrophages were cultured in phenol red-free DMEM with high glucose (4500 mg/L) and L-Glutamine (4 mM/L) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (10,000U/mL), and streptomycin (10,000g/mL). The cells were maintained in a humidied incubator containing 5% CO2 at 37C. For all experiments, cells were grown to 80%90% conuence, and subjected to no more than 20 cell passages. Cells were scraped out from the plastic culture asks then centrifuged at 110g at 4C for 10 minutes. The medium was then removed and the cells were suspended with fresh DMEM containing the same supplements. The concentration was adjusted to 2 106 cells/mL and cell viability was always more than 80%, as determined using a standard trypan blue cellcounting technique. Cells were dispensed (50L) into wells

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Cytoprotective and anti-inflammatory activities of liposomal piroxicam

of tissue culture-grade 96-well plates (ie, 1105 cells/well) and incubated for 2hours at 37C in 5% CO2 atmosphere to attach the cells. Unattached cells were discarded gently after 2hours. The attached cells were then stimulated with 10 g/mL E. coli LPS in the presence or absence of the treatment sample (ie, piroxicam or liposome-encapsulated piroxicam) at a nal volume of 800 L/well. The nal concentration of DMSO in each well, including in the nonstimulated and non-treated control cells, was maintained at 0.67%. Cells were then incubated for 24hours at 37C in a humidied 5% CO2 atmosphere.

treatment samples to inhibit NO2 below the level produced by control cells.

Inflammatory cytokine immunoassay

To determine the effects of treatment samples on the production of inammatory cytokines (ie, TNF-, IL-1, and IL-10), cells were cultured and treated as described in the previous section. Supernatants collected from wells of a 96-well plate were assayed using specic enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientic) in accordance with the manufacturers instructions.

Measurement of cell viability

Following overnight incubation with treatment samples, cell viability was assessed by MTT assay after the removal of spent media from the 96-well plates. Cleavage of the MTT ring only occurs in the active mitochondria of living cells, hence, this assay was based on the ability of viable cells to reduce MTT from a pale yellow water-soluble dye to a dark blue insoluble formazan product.22 In brief, MTT was rst dissolved in phosphate-buffered saline at 5 mg/mL. Thereafter, the stock MTT solution was ltered to sterilize and remove any insoluble residue before being kept away from direct light exposure. Stock MTT solution (10 L per 100 L medium) was added to all wells of the assay plates, and these were incubated at 37C. After 4hours, the medium was removed and the remaining dark blue MTT crystals in each well were fully dissolved by the addition of 100L DMSO. Absorbance of each well was measured using a microplate reader (Innte M200, Tecan, Grdig, Austria) at a test wavelength of 570nm. Cell viability was determined as the relative reduction of optical density, which correlates with the amount of viable cells in relation to the control cells.

Prostaglandin E2 (PGE2) immunoassay

The concentration of PGE2 produced from endogenous arachidonic acid in the macrophage cell cultures, which were initially treated with different treatment samples, was measured. The collected cell culture supernatants were quantied by specic ELISA kit (Thermo Fisher Scientic) according to the manufacturers instructions.

Statistical analyses

Data reported are presented as the mean standard error of the mean of triplicate cultures of three independent experiments (n=9). Statistical signicance was determined using analysis of variance followed by Dunnetts multiple comparison test among groups, and Students t-test for comparison between means of two groups. P values , 0.05 were considered indicative of signicance. All statistical analyses were carried out using SPSS software (v 16.0; IBM, Armonk, NY, USA).

Results Characterization of liposomal samples

Table1shows the entrapment and size proles of the liposomal samples used in the experimental work reported here. The use of a previously optimized proliposome method for piroxicam encapsulation successfully resulted in drug-loaded liposomes with satisfactory drug entrapment proles. All samples (ie, blank and drug-loaded liposomes) had sufciently small particle sizes in the nanometer range and exhibited a relatively narrow size distribution. These spontaneously formed liposomal sample particles were spherical and were seen to have concentric lamellae under transmission electron microscope (Figure1).

Measurement of NO/nitrite production

The level of nitrite (NO2) in cell-free culture supernatants, which reects intracellular NO synthase activity, was determined by Griess reaction.23 Briey, 100 L of supernatant was collected from each of the stimulated and treated cell cultures. These supernatants were mixed with an equal volume of Griess reagent in a 96-well plate and incubated at room temperature for 10minutes. This was followed by spectrophotometric measurements using a microplate reader at a test wavelength of 550nm. The NO2 concentrations in the supernatants were determined through a comparison with a sodium nitrite standard curve. Percentage inhibition was calculated based on the ability of the different

MTT assay
Treatment-induced cytotoxic responses in RAW 264.7macrophages, expressed as cell viability, are presented in Table2.

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Table 1 Entrapment and size profiles of liposomal samples

Liposomal sample Entrapment profile Entrapment capacity (g piroxicam/g Pro-lipo) Piroxicam-loaded liposomes Blank liposomes 805.5 43.9 N/A Percent entrapped (%) 15.2 1.2 N/A Size profile Particle size (nm) 362.6 15.4 376.3 7.8 Polydispersity index 0.453 0.009 0.455 0.005

Note: Values shown are mean standard error of the mean (n = 6). Abbreviations: g, micrograms; N/A, not applicable; nm, nanometers.

The cell viability of the basal group (ie, non-treated and non-stimulated cells) was designated 100%, indicating no cytotoxicity. Data obtained show that 24-hour exposure of nonLPS-stimulated cells to different treatment samples, except the highest dose of piroxicam (ie, 0.4mg/mL), did not cause signicant decrement in cell viability. Contrarily, simultaneous LPS-stimulation resulted in signicant cytotoxic activities in cells treated with piroxicam and liposome-encapsulated A

piroxicam at drug dosages of 0.1, 0.2, and 0.4mg/mL. Only the highest dose of piroxicam showed a signicant difference in cell viability when statistically compared with respective control groups. Further analyses revealed that there was a signicant difference between 0.4mg/mL piroxicam and its equivalent drug dosage of liposome-encapsulated piroxicam in non-LPS-stimulated cells. This indicates that the latter exhibited a lower cytotoxic effect than the former.

200 nm

200 nm

Figure1 Transmission electron microscope photographs of (A) blank liposomes and (B) piroxicam-loaded liposomes.

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Cytoprotective and anti-inflammatory activities of liposomal piroxicam

Table 2 Effects of different treatments on the cell viability of RAW 264.7 macrophages
Treatment group Basal Piroxicam Drug dosage (mg/mL) 0.0 0.0 (control) 0.1 0.2 0.4 0.0 0.1 0.2 0.4 Cell viability (%) Non-stimulated cells 100.00 2.34 98.87 1.43 98.12 2.08 94.10 1.93 91.62 1.23a,b 99.14 1.97 98.59 2.00 96.66 1.81 95.98 1.53c LPS-stimulated cells N/A 94.84 2.00 92.17 1.67a 91.98 1.87a 86.75 1.53a,b 96.22 1.75 92.78 2.05a 92.04 1.33a 90.22 1.95a

Liposome-encapsulated piroxicam

Notes: Values shown are mean standard error of the mean. aSignificant difference (P , 0.05) when compared to basal; bsignificant difference (P , 0.05) when compared to control (piroxicam; 0 mg/mL); csignificant difference (P , 0.05) when compared to group with equivalent dosage of piroxicam. Abbreviations: LPS, lipopolysaccharide; mg/mL, milligrams per milliliter.

NO assay
The effect of the different treatments on NO production in RAW 264.7 macrophages is presented in Figure 2. All treatment groups in the present study, regardless of drug dosage or liposomal encapsulation, exhibited signicantly less LPS-induced NO production when compared with the control group. In addition, statistical analyses also show that liposome-encapsulated piroxicam exhibited signicantly greater NO reduction when compared with its non-encapsulated form at all equivalent drug dosages. The percentage of inhibition at 0.1, 0.2, and 0.4 mg/mL was increased by 26.75%, 28.70%, and 11.32%, respectively.
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Production of inflammatory cytokines

The concentrations of TNF- and IL-1 (indicative of proinammatory cytokine activity), as well as IL-10 (indicative of anti-inammatory activity) in the treated RAW 264.7macrophages are illustrated in Figures35, respectively. Results show that concurrent piroxicam treatment with these cells signicantly inhibited the production of both TNF- and IL-1 at higher dosages (ie, 0.2 and 0.4mg/mL). In contrast, liposome-encapsulated piroxicam signicantly inhibited the LPS-induced rise of both pro-inammatory cytokines at a dosage as low as 0.1mg/mL. Further statistical analyses conrmed that liposomal piroxicam formulations were

20

,# ,#

Nitric oxide (M)

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0.2 mg/mL

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Liposome-encapsulated piroxicam

Figure2 Effects of different treatment upon nitric oxide (NO) production. Notes: Values shown are mean standard error of the mean (n=9/group). *Significant difference (P,0.05) when compared to control (piroxicam; 0mg/mL); #significant difference (P,0.05) when compared to group with equivalent dosage of piroxicam.

0 mg/mL

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TNF- (pg/mL)

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Piroxicam

Liposome-encapsulated piroxicam

Figure3 Effects of different treatment upon tumor necrosis factor-alpha (TNF-) production. Notes: Values shown are mean standard error of the mean (n=9/group). *Significant difference (P,0.05) when compared to control (piroxicam; 0mg/mL); #significant difference (P,0.05) when compared to group with equivalent dosage of piroxicam.

signicantly more effective in inhibiting pro-inammation cytokines than their equivalent piroxicam doses. The percentage of TNF- inhibition was increased by 11.58%, 13.29%, and 10.69% at drug doses of 0.1, 0.2, and 0.4mg/mL, respectively. The highest augmentation of IL-1 inhibition, which resulted
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from liposomal encapsulation, was exhibited at a drug dose of 0.2mg/mL. The inhibition increased by as much as 26.66%. Liposome-encapsulated piroxicam, at the highest tested dosage (ie, 0.4mg/mL) also successfully reversed the LPS-induced rise of IL-1 in the stimulated macrophages.

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IL-1 (pg/mL)

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Figure4 Effects of different treatment on interleukin (IL)-1 production. Notes: Values shown are mean standard error of the mean (n=9/group). *Significant difference (P,0.05) when compared to control (piroxicam; 0mg/mL); #significant difference (P,0.05) when compared to group with equivalent dosage of piroxicam.

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0.4 mg/mL

0 0 mg/mL

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Cytoprotective and anti-inflammatory activities of liposomal piroxicam

IL-10 (pg/mL)

4 3 2 1 0 0 mg/mL Basal

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Piroxicam

Liposome-encapsulated piroxicam

Figure5 Effects of different treatment on interleukin (IL)-10 production. Notes: Values shown are mean standard error of the mean (n=9/group). *Significant difference (P,0.05) when compared to control (piroxicam; 0mg/mL); #significant difference (P,0.05) when compared to group with equivalent dosage of piroxicam.

In the case of anti-inammatory cytokine activity, treatments potentiated a signicant rise in IL-10 concentration in the RAW 264.7macrophages. Further, signicantly higher cytokine concentrations compared with the control group were exclusively exhibited by liposomeencapsulated piroxicam at 0.2 and 0.4mg/mL. The IL-10 concentrations when the liposome-encapsulated piroxicam concentration was 0.2 and 0.4 mg/mL were 3.21% and 4.70%, respectively, higher than their equivalent dosages of piroxicam.

Discussion
Macrophages are particularly important for both innate and adaptive immunity due to their crucial role in many inammatory processes.24,25 These cells have been implicated in many disease states, including inammation, infection, atherosclerosis, diabetes, lysosomal storage disease, lupus, and cancer.26 In this research, RAW 264.7 (which is a macrophage-like, Abelson murine leukemia virus-transformed cell line derived from BALB/c mice) was selected to study the events that regulate the function of diseased cells.3,26 Using this wellestablished cellular model of inammation, the main aim of the present experimental work was to evaluate the potential of a liposomal drug-encapsulation strategy in improving the safety and anti-inammatory properties of piroxicam. Similar to other NSAIDs, piroxicam has been associated with diverse in vivo drug-induced toxicities. 18 The inherent cytotoxic effects of different treatment on the cellular model used in the present study were predetermined and compared using MTT assay. High percentages of cell viability ($86.75%) were observed for all treatment groups in the present work, indicating that the treatments induced negligible cytotoxic effects on RAW 264.7 macrophages. Data analyses indicate that blank liposomes were not toxic under the present experimental conditions. The results also show that piroxicam, whether non-encapsulated or in liposome-encapsulated form, synergistically acted with

PGE2 biosynthesis

The production of PGE2, a well-known inammatory mediator regulated by piroxicam, is presented in Figure6. Results show a dose-dependent inhibition of PGE2 biosynthesis by both non-encapsulated and liposome-encapsulated forms of piroxicam at 0.1, 0.2, and 0.4mg/mL. Statistical analyses also revealed that the LPS-induced rise in PGE2 was successfully reversed on concurrent treatment with piroxicam at 0.4mg/mL, as well as the liposome-encapsulated drug at 0.2 and 0.4mg/mL, with inhibition between 93.98% and 98.94%. Further analysis revealed that the PGE2 inhibitory activities of liposome-encapsulated piroxicam at 0.2 and 0.4mg/mL were signicantly greater than their equivalent drug dosages of piroxicam. The percentage of inhibition was increased by as much as 18.91% for 0.2mg/mL.

0.4 mg/mL

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PGE2 (pg/mL)

6000 5000 4000 3000 2000 1000 0 0 mg/mL Basal

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,#
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Piroxicam

Liposome-encapsulated piroxicam

Figure6 Effects of different treatment on prostaglandin E2 (PGE2) biosynthesis. Notes: Values shown are mean standard error of the mean (n=9/group). *Significant difference (P,0.05) when compared to control (piroxicam; 0mg/mL); #significant difference (P,0.05) when compared to group with equivalent dosage of piroxicam.

bacterial LPS endotoxin to cause a signicant reduction in cell viability in the macrophages. Nevertheless, at all equivalent dosages, there was no statistically signicant difference between piroxicam and liposome-encapsulated piroxicam. Interestingly, liposomal encapsulation of the drug resulted in relatively higher percentages of cell viability, particularly at high drug dosages in non-stimulated cells. These ndings suggest that the strategy of encapsulating the drug within liposomes possesses a protective effect in reducing the toxicity of piroxicam. More work (eg, acute and chronic in vivo toxicity testing) is required to further support this claim. The effects of endotoxin-induced injury on macrophages are extensive. They include a decreased capacity to produce antigen, as well as altered production of key mediators such as free radicals, reactive oxygen species, cytokines, and bioactive lipids.8,17,24 Nowadays, the treatment for inammatory diseases is largely based on interrupting the action or synthesis of critical mediators that drive the hosts response to injury. Various therapeutic agents, including NSAIDs, steroids, and antihistamines, have been developed for this purpose. 1 In the present work, the efcacy of different piroxicam formulations in modulating the production of inammatory mediators was evaluated using LPS-stimulated cells. Data obtained from the assays undertaken in this study prove that bacterial LPS endotoxin triggered signicant production of NO,

inammatory cytokines (ie, TNF- and IL-1), and PGE2 in RAW 264.7macrophages. NO, a highly reactive free radical produced from L-arginine by NO synthase (NOS), is well known for its involvement in diverse physiological and pathological processes.8,27 Under normal physiological conditions, the production of this short-lived molecular messenger is regulated by constitutive isoforms of NOS (ie, neuronal and endothelial NOS). This minute quantity of NO plays a benecial role as a smooth muscle vasodilator; a neurotransmitter; and in nonspecic immune responses to infection, host defense, and cytotoxicity.2830 However, on exposure to specic stimulants such as bacterial LPS endotoxin, inammatory cytokines, or calcium ionophores, an enzyme known as inducible NOS results in prolonged and high-output NO production. Excessive production of NO can be harmful and result in inammation, intracellular oxidative stress, and autoimmune diseases. Thus, the degree of inducible NOS or total NO suppression provides a measure to assess the efcacy of a particular treatment in inhibiting an inammatory process.3134 In the present study, the data obtained during NO assay indicate that all treatment groups exhibited good NO-inhibitory activities in LPS-stimulated macrophages. Further statistical analyses revealed that each group of liposome-encapsulated piroxicam possessed signicantly stronger NO inhibition than their respective equivalent dosage of piroxicam. Since the improved inhibitory activities were not due to cytotoxic

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Cytoprotective and anti-inflammatory activities of liposomal piroxicam

effects, as indicated by cell viability analyses, this study has proven that liposomal encapsulation technology enhances NO-induced inammatory processes. During inammation, a distinct cytokine cascade unfolds. Copious amounts of TNF- are released as a response to the invasive stimuli. In turn, the over-production of TNF- stimulates the release of various inammatory mediators such as IL-1, IL-6, NO, and PGE2. These modulate important cellular events such as gene expression, DNA damage, and cellular proliferation, which lead to aggravation and progression of various diseases.24,35 Thus, cellular manipulation for synthesis of cytokines (eg, TNF and IL-1) is important in regulating inammatory responses. Results from our study demonstrate that liposome-encapsulated piroxicam, at lower drug dosages, is sufcient to inhibit the formation of TNF- and IL-1. Further data analyses also support the fact that liposomal encapsulation strategy is effective in improving the downregulation of pro-inammatory cytokines without needing to increase the drug dosage. In contrast to the pro-inammatory cytokines, in the present work, IL-10 formation was dose-dependently increased by liposome-encapsulated piroxicam. IL-10 is an anti-inammatory cytokine produced by monocytes and lymphocytes. It has been reported to inhibit the production of pro-inammatory cytokines including TNF-. Additionally, IL-10might exert its anti-inammatory action by inducing the formation of the IL-1 receptor antagonist.36,37 Thus, the present experimental ndings suggest that the liposomes resulted in attenuation of the pro-inammatory/ anti-inammatory cytokines ratio, which in turn contributed to the improved anti-inammatory efcacy of the liposomeencapsulated drug samples. Apart from modulating NO and cytokine release, various anti-inammatory drugs including piroxicam are also able to inhibit PG synthesis. PGs, in particular PGE2, are molecules with potent inflammatory and vasodilatatory functions. These important prostanoids are involved in a wide variety of physiological and pathological processes.6,25 There are two isoforms of cyclooxygenase (COX), the key enzyme for the conversion of arachidonic acids to PGs. COX-1 is constitutively expressed in many normal tissues, whereas COX-2 is an inducible enzyme involved primarily in the regulation of inammatory and immunological events. Expression of COX-2 is signicantly upregulated by inammatory stimuli (eg, bacterial LPS endotoxin, growth factors, cytokines, oncogenes, and carcinogens), which produces PGs, predominantly PGE2, that contribute to pain and swelling in trauma and in inammatory and malignant diseases.11,29,32 Thus, reduction

in the level of COX-mediated PGE2 synthesis is an effective strategy to inhibit inammation as well as carcinogenesis. Compared with that of macrophages treated with LPS alone, in our study, both piroxicam and liposomeencapsulated piroxicam resulted in the signicant inhibition of PGE2 synthesis. However, the dose-dependent inhibitory activities were signicantly stronger in macrophages treated with liposome-encapsulated drug samples. Results show that, on treatment with moderate and high dosages of liposomeencapsulated piroxicam, PGE2 accumulation in macrophages was signicantly lower than with the respective equivalent dosages of free piroxicam. This signies that the modulation of PGE2 secretion during an inammatory process was more effective using the liposome-encapsulated drug.

Conclusion
The present ndings reveal that both piroxicam and liposomeencapsulated piroxicam exhibit different degrees of cytotoxic and inammatory responses in RAW 264.7 macrophages. In summary, the cell viability study shows that liposomes protect macrophages from drug-induced and LPSinduced cytotoxicity. Results also demonstrate that the liposome-encapsulated piroxicam had stronger in vitro anti-inammatory activities than the non-encapsulated form of piroxicam. Signicantly reduced production of major pro-inammatory mediators such as NO, TNF-, IL-1, and PGE2 was observed in macrophages treated with these liposome-encapsulated samples. Additionally, using liposomal drug-encapsulation technology, a lower drug dose was sufcient to inhibit the production of TNF- and IL-1 in LPS-stimulated macrophages. Finally, the present work indicates that the production of the anti-inammatory cytokine IL-10may also contribute to the improved therapeutic effects of liposome-encapsulated piroxicam.

Acknowledgment
Funding for this work was provided by a research grant from the Ministry of Higher Education, Malaysia (Grant no 04-1007-277FR).

Disclosure
The authors report no conicts of interest in this work.

References

1. Gayathri B, Manjula N, Vinaykumar KS, Lakshmi BS, Balakrishnan A. Pure compound from Boswellia serrata extract exhibits anti-inammatory property in human PBMCs and mouse macrophages through inhibition of TNFalpha, IL-1beta, NO and MAP kinases. Int Immunopharmacol. 2007;7(4):473482.

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Chiong etal 2. Kim HG, Shrestha B, Lim SY, et al. Cordycepin inhibits lipopolysaccharide-induced inammation by the suppression of NFkappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells. Eur J Pharmacol. 2006;545(23):192199. 3. Hsueh R, Roach T. Passage Procedure for RAW 264.7 cells. AfCS Procedure Protocol PP00000159. Dallas: Alliance for Cellular Signaling; 2003. Available from: http://www.signaling-gateway.org/data/cgi-bin/ ProtocolFile.cgi/afcs_PP00000159.pdf?pid=PP00000159. Accessed February 13, 2013. 4. Orlicek SL, Meals E, English BK. Differential effects of tyrosine kinase inhibitors on tumor necrosis factor and nitric oxide production by murine macrophages. J Infect Dis. 1996;174(3):638642. 5. Harasstani OA, Moin S, Tham CL, etal. Flavonoid combinations cause synergistic inhibition of proinammatory mediator secretion from lipopolysaccharide-induced RAW 264.7 cells. Inamm Res. 2010; 59(9):711721. 6. Kim YK, Kang HJ, Lee KT, Choi JG, Chung SH. Anti-inammation activity of Actinidia polygama. Arch Pharm Res. 2003;26(12):10611066. 7. Reddy MC, Subhashini J, Mahipal SV , etal. C-Phycocyanin, a selective cyclooxygenase-2inhibitor, induces apoptosis in lipopolysaccharidestimulated RAW 264.7macrophages. Biochem Biophys Res Commun. 2003;304(2):385392. 8. Kim ID, Ha BJ. Paeoniorin protects RAW 264.7macrophages from LPS-induced cytotoxicity and genotoxicity. Toxicol In Vitro. 2009; 23(6):10141019. 9. Jalava PI, Salonen RO, Pennanen AS, etal. Effects of solubility of urban air ne and coarse particles on cytotoxic and inammatory responses in RAW 264.7macrophage cell line. Toxicol Appl Pharm. 2008;229(2): 146160. 10. Israf DA, Tham CL, Syahida A, etal. Atrovirinone inhibits proinam matory mediator synthesis through disruption of NF-kappaB nuclear translocation and MAPK phosphorylation in the murine monocytic macrophage RAW 264.7. Phytomedicine. 2010;17(10):732739. 11. Verdina A, Cardillo I, Nebbioso A, et al. Molecular analysis of the effects of Piroxicam and Cisplatin on mesothelioma cells growth and viability. J Transl Med. 2008;6:27. 12. Vasudevan DM, Sreekumari S, Vaidyanathan K. Chemical basis of life. In: Textbook of Biochemistry for Medical Students, 6th ed. New Delhi: Jaypee Brothers Medical; 2011:182. 13. Lopes LB, Scarpa MV, Pereira NL, Oliveira LC, Oliveira AG. Interaction of sodium diclofenac with freeze-dried soya phosphatidylcholine and unilamellar liposomes. Revista Brasileira de Cincias Farmacuticas. 2006;42(4):497504. 14. Chen H, Khemtong C, Yang X, Chang X, Gao J. Nanonization strategies for poorly water-soluble drugs. Drug Discov Today. 2011; 16(78):354360. 15. Fricker G, Kromp T, Wendel A, etal. Phospholipids and lipid-based for mulations in oral drug delivery. Pharm Res. 2010;27(8):14691486. 16. Chakraborty S, Shukla D, Mishra B, Singh S. Lipid an emerging platform for oral delivery of drugs with poor bioavailability. Eur J Pharm Biopharm. 2009;73(1):115. 17. Hussein H, Hussain H, Salbiah MS, Muhaini O, Low YL, Beh PK. Use of drugs for rheumatological and bone disorders. In: Faridah AM, Sivasampu S, Lian LM, Hazimah H, Kok LC, Chinniah RJ, editors. Malaysian Statistics on Medicines 2007. Kuala Lumpur: Ministry of Health Malaysia; 2010:109116. 18. Mosby. Piroxicam. In: 2007 Mosbys Drug Consult, 17th ed. St Louis, MO: Elsevier; 2006:II2336II2338. 19. Leikin JB, Paloucek FP. Piroxicam. In: Poisoning and Toxicology Handbook, 4th ed. Boca Raton, FL: CRC Press; 2008:553554.

Dovepress 20. Chiong HS, Nazrul Hakim M, Sulaiman MR, etal. Development and characterisation study of liposomes-encapsulated piroxicam. Int J Drug Deliv. 2011;3(1):6473. 21. Chiong HS, Somchit MN, Azhar Y, Ong SG, Yuen KH. Preparation and in-vitro evaluation of blank and piroxicam-loaded liposomes. J Ind Tech. 2010;19(1):1730. 22. Israf DA, Khaizurin TA, Syahida A, Lajis NH, Khozirah S. Cardamonin inhibits COX and iNOS expression via inhibition of p65NF-kappaB nuclear translocation and Ikappa-B phosphorylation in RAW 264.7macrophage cells. Mol Immunol. 2007;44(5):673679. 23. Park KS, Kim BH, Chang IM. Inhibitory Potencies of Several Iridoids on Cyclooxygenase-1, Cyclooxygnase-2 Enzymes Activities, Tumor Necrosis factor- and Nitric Oxide Production In Vitro. Evid Based Complement Alternat Med. 2010;7(1):4145. 24. Chou TC, Fu E, Shen EC. Chitosan inhibits prostaglandin E2 formation and cyclooxygenase-2 induction in lipopolysaccharide-treated RAW 264.7 macrophages. Biochem Biophys Res Commun. 2003;308(2): 403407. 25. Sun LK, Wahl P, Bilic G, Wthrich RP. CD44-mediated cyclooxygenase-2 expression and thromboxane A2 production in RAW 264.7macrophages. Inamm Res. 2001;50(10):496499. 26. Genlantis. A Highly Efcient Method of Transfecting RAW 264.7 Cells. San Diego, CA: Genlantis; 2009. 27. Lee J, Kim KA, Jeong S, et al. Anti-inammatory, anti-nociceptive, and anti-psychiatric effects by the rhizomes of Alpinia ofcinarum on complete Freunds adjuvant-induced arthritis in rats. J Ethnopharmacol. 2009;126(2):258264. 28. Kumar-Roin S, Matsui M, Reybier K, etal. Ability of certain plant extracts traditionally used to treat ciguatera sh poisoning to inhibit nitric oxide production in RAW 264.7macrophages. J Ethnopharmacol. 2009;123(3):369377. 29. Yen GC, Duh PD, Huang DW, Hsu CL, Fu TY. Protective effect of pine (Pinus morrisonicola Hay.) needle on LDL oxidation and its antiinammatory action by modulation of iNOS and COX-2 expression in LPS-stimulated RAW 264.7 macrophages. Food Chem Toxicol. 2008;46(1):175185. 30. Garcia X, Stein F. Nitric oxide. Semin Pediatr Infect Dis. 2006;17(2): 5557. 31. Ahn EK, Jeon HJ, Lim EJ, Jung HJ, Park EH. Anti-inammatory and anti-angiogenic activities of Gastrodia elata Blume. J Ethnopharmacol. 2007;110(3):476482. 32. Kim KW, Ha KT, Park CS, etal. Polygonum cuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2gene expressions in RAW 264.7macrophages. Vascul Pharmacol. 2007;47(23):99107. 33. Lee MH, Lee JM, Jun SH, etal. The anti-inammatory effects of Pyro lae herba extract through the inhibition of the expression of inducible nitric oxide synthase (iNOS) and NO production. J Ethnopharmacol. 2007;112(1):4954. 34. Sharma JN, Al-Omran A, Parvathy SS. Role of nitric oxide in inam matory diseases. Inammopharmacology. 2007;15(6):252259. 35. Loram LC, Fuller A, Fick LG, Cartmell T, Poole S, Mitchell D. Cytokine proles during carrageenan-induced inammatory hyperalgesia in rat muscle and hind paw. J Pain. 2007;8(2):127136. 36. Saraiva M, OGarra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol. 2010;10(3):170181. 37. Li L, Rossoni G, Sparatore A, Lee LC, Del Soldato P, Moore PK. Anti-inammatory and gastrointestinal effects of a novel diclofenac derivative. Free Radic Biol Med. 2007;42(5):706719.

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